The development of tolerance to opioids limits their effectiveness for the treatment of pain. A wide variety of opioids that are commonly used in the clinic for the treatment of pain and drug addiction have dramatically different pharmacological properties. One primary difference between agonists is the ability of different agonist to cause varying amounts of desensitization and internalization of mu opioid receptors. Agonists fall into three major groups, those that induce both desensitization and internalization, those that induce desensitization but not internalization and those that are ineffective at both. The role that desensitization and internalization have in the development to tolerance and dependence to opioids has been a controversial subject that has been studied in a variety of model systems. This exploratory proposal will develop the method of Flurescence Correlation Spectroscopy to investigate the mechanism that accounts for the varying actions of different opioid agonists on the mu opioid receptor. This is a relatively new method that uses optical measurements of the mobility of fluorescent molecules within a very small volume. These measurements are made at the plasma membrane, within intracellular compartments in various parts of the cell, including the cell body, dendrites, axons and terminals. The mobility of molecular is directly related to the molecular interactions such that agonist/receptor and receptor/effector associations will be identified. This study will use agonists that are fluorescent (aim 1) and fluorencescently tagged mu-opioid receptors (aim 2) in a model system, HEK293 cells that stably express epitope-tagged mu-opioid receptors. Electrophysiological, biochemical and imaging methods have been used to characterize many of the steps in opioid-receptor dependent signaling. This exploratory proposal will introduce a new way to investigate the mechanisms that underlie the different pharmacological profiles of opioid agonists. In the 2 years afforded this proposal, this method will be developed using a very well characterized model, the HEK293 cells. Once this method is established, the ultimate goal of applying it to study of primary neurons will be pursued. By gaining knowledge of the processes that are selectively affected by some agonists and not others, the mechanisms underlying the development of tolerance and dependence may be identified and applied to more effective treatment of chronic pain and addiction.